Pituitary lutropin hormone (LH) is suppressed during the luteal phase of the menstrual cycle by negative feedback from ovarian steroids (Leung et al., “Interactions of Steroids and Gonadotropins in the Control of Steroidogenesis in the Ovarian Follicle,” Annu Rev Physiol 42:71-82 (1980); Christenson et al., “Maturation of Ovarian Follicles in the Prepubertal Gilt,” J Reprod Fertil Suppl 33:21-36 (1985) and Abraham et al., “Simultaneous Radioimmunoassay of Plasma FSH, LH, Progesterone, 17-Hydroxyprogesterone, and Estradiol-17beta During the Menstrual Cycle,” J Clin Endocrinol 34:312-318 (1972)). Human chorionic gonadotropin (hCG), a placental hormone produced in the female at the time of implantation, provides a backup for pituitary LH to rescue the corpus luteum of pregnancy. hCG is a heterodimeric glycoprotein hormone consisting of an α and a β subunit that stimulates intracellular levels of cAMP via a G-protein-coupled receptor. A distinct feature of the hCG-β subunit is the presence of a C-terminal extension (CTP) of 24 amino acids, which produce specific antibodies to hCG-β with little cross-reaction with LH. In a gonadal cell, hCG binds to its cell surface receptor (LH/CG-R), resulting in an increase in the concentration of intracellular cAMP (Wu et al., “Protein Engineering of Novel Constitutively Active Hormone-Receptor Complex,” J Biol Chem 271:31638-31642 (1996). hCG and hLH (human LH) are identical in structure except for the CTP of the β-subunit of hCG, which is highly glycosylated and has three serine-linked carbohydrate moieties (Cole et al., “The Structures of the Serine-Linked Sugar Chains on Human Chorionic Gonadotropin,” Biochem Biophys Res Commun 126 (1):333-339 (1985)). hLH and hCG, by virtue of their structural similarity, bind to the same lutropin receptor (LH-R) in the gonads. The LH-R is also a member of the G protein-coupled receptor (GPCR) family and contains a relatively large, highly glycosylated, N-terminal extracellular domain (ECD) known for high affinity ligand binding (Xie et al., “Extracellular Domain of Lutropin/Choriogonadotropin Receptor Expressed in Transfected Cells Binds Choriogonadotropin with High Affinity,” J Biol Chem 265(35):21411-21414 (1990) and Ji et al., “Exons 1-10 of the Rat LH Receptor Encode a High Affinity Hormone Binding Site and Exon 11 Encodes G-Protein Modulation and a Potential Second Hormone Binding Site,” Endocrinology 128:2648-2650 (1991)), a seven-transmembrane domain, and a short intracellular cytoplasmic tail (McFarland et al., “Lutropin-Choriogonadotropin Receptor: An Unusual Member of the G Protein-Coupled Receptor Family,” Science 245(4917):494-499 (1989)). The extracellular domain is characterized by a motif of imperfect leucine-rich repeats, which contributes largely to the high affinity hormone binding of the receptor (Xie et al., “Extracellular Domain of Lutropin/Choriogonadotropin Receptor Expressed in Transfected Cells Binds Choriogonadotropin with High Affinity,” J Biol Chem 265(35):21411-21414 (1990)). cDNAs of LH receptor have considerable interspecies homology among vertebrates (Ascoli et al., The Lutropin/Choriogonadotropin Receptor, A 2002 Perspective,” Endocr Rev 23(2):141-174 (2002)). hLH-R and hCG-β are also antigenic at the interspecies level (Ascoli et al., The Lutropin/Choriogonadotropin Receptor, A 2002 Perspective,” Endocr Rev 23(2):141-174 (2002); Pal et al., “Active Immunization of Baboons (Papio anubis) with the Bovine LH Receptor,” J Reprod Immunol 21(2):163-174 (1992); Pal et al., “Biological Actions of Monoclonal Antibodies to Bovine Lutropin Receptor,” J Reprod Immunol 22(1):103 (1992); Remy et al., “Immunization Against Exon 1 Decapeptides From the Lutropin/Choriogonadotropin Receptor or the Follitropin Receptor as Potential Male Contraceptive,” J Reprod Immunol 32(1):37-54 (1996); Singh et al., “Effect of Immunization with Lutropin-Receptor on the Ovarian Function of Rabbits,” J Immunoassay 16(1):1-16 (1995), Vaitukaitis et al., “A Radioimmunoassay Which Specifically Measures Human Chorionic Gonadotropin in the Presence of Human Luteinizing Hormone,” Am J Obstet Gynecol 113(6):751-758 (1972)). Hence, hLH-R and hCG, and their functional epitopes, provide vital targets to be manipulated by genetic engineering to produce unique anti-fertility antigens.
Efficacies of antibodies to the hLH-R and hCG-β individually have been amply demonstrated in the regulation of gonadal function. There are a number of reports that qualify LH-R as a potential anti-fertility antigen (Remy et al., “Immunization Against Exon 1 Decapeptides From the Lutropin/Choriogonadotropin Receptor or the Follitropin Receptor as Potential Male Contraceptive,” J Reprod Immunol 32(1):37-54 (1996); Saxena et al., “Modulation of Ovarian Function in Female Dogs Immunized With Bovine Luteinizing Hormone Receptor,” Reprod Domest Anim 37(1):9-17 (2002), and Saxena et al., “Effect of Immunization with Bovine Luteinizing Hormone Receptor on Ovarian Function in Cats,” Am J Vet Res 64(3):292-298 (2003)). The injection of the recombinant mouse LH-R with hormone binding region to male mice induced immunity against the receptor. The data indicated that the specific anti-gonadotropin receptor vaccination could potentially be used as a fertility control procedure in males (Remy et al., “Immunization Against Exon 1 Decapeptides From the Lutropin/Choriogonadotropin Receptor or the Follitropin Receptor as Potential Male Contraceptive,” J Reprod Immunol 32(l):37-54 (1996)). Previous studies showed that actively immunizing female dogs and cats with highly purified bovine LH-R can produce antibodies to the LH-R, which suppresses progesterone synthesis due to the blockade of the gonadal receptor (Saxena et al., “Modulation of Ovarian Function in Female Dogs Immunized With Bovine Luteinizing Hormone Receptor,” Reprod Domest Anim 37(1):9-17 (2002) and Saxena et al., “Effect of Immunization with Bovine Luteinizing Hormone Receptor on Ovarian Function in Cats,” Am J Vet Res 64(3):292-298 (2003)).
Elucidation of the human genome has provided an impetus for genetic engineering, gene therapy, and stem cell research. Recently, gene based expression of functional recombinant proteins and their epitopes as well as antibodies against them have permitted the topographical mapping of receptors to identify active sites and understand the mechanism of ligand receptor binding. Synthesis of chimeric genes composed of desired functional epitopes of more than one gene to produce recombinant proteins has become possible (Sugahara et al., “Biosynthesis of a Biologically Active Single Chain Containing the Human Common a and Chorionic Gonadotropin β Subunits in Tandem,” Proc Natl Acad Sci USA 92:2041-2045 (1995); Sugahara et al., “Expression of Biologically Active Fusion Genes Encoding the Common a Subunit and Either the CGβ of FSHβ Subunits: Role of a Linker Sequence,” Mol Cell Endocrinol 125:71-77 (1996); and Heikoop et al., “Structure-Based Design and Protein Engineering of Intersubunit Disulfide Bonds in Gonadotropins,” Nat Biotechnol 15(7):658-662 (1997); Heikoop et al., “Evaluation of Subunit Truncation and the Nature of the Spacer for Single Chain Human Gonadotropins,” Eur J Biochem 245(3):656-662 (1997)). A single peptide chain containing α and β subunits of human chorionic gonadotropin (hCG) in tandem has been shown to exhibit biological activity (Sugahara et al., “Biosynthesis of a Biologically Active Single Chain Containing the Human Common α and Chorionic Gonadotropin β Subunits in Tandem,” Proc Natl Acad Sci USA 92:2041-2045 (1995)). Fusion genes encoding the common α-subunit of hCG and FSH-β subunit also expressed a biologically active protein (Sugahara et al., “Expression of Biologically Active Fusion Genes Encoding the Common α Subunit and Either the CGβ of FSH-β Subunits: Role of a Linker Sequence,” Mol Cell Endocrinol 125:71-77 (1996)). Narayan et al., “Yoked Complexes of Human Choriogonadotropin and the Lutropin Receptor: Evidence that Monomeric Individual Subunits are Inactive,” Mol Endocrinol 16(12):2733-2745 (2002), constructed two (hCG)-LHR complexes where the two hormone subunits and the receptor were engineered to form single polypeptide chains.
Chemical conjugates to create functional chimeric proteins have been made to enhance their immunogenicity and/or to modulate metabolic clearance rates and biological activity (Klein et al., “Pharmacokinetics and Pharmacodynamics of Single-Chain Recombinant Human Follicle-Stimulating Hormone Containing the Human Chorionic Gonadotropin Carboxyterminal Peptide in the Rhesus Monkey,” Fertil Steril 77(6):1248-1255 (2002)). Chimeric proteins have also been produced where the common human α-subunit of human glycoprotein hormone has been non-covalently linked to the hormone specific β-subunits of hCG and hFSH to express respective intact hormones, and their ability to interact with LH-hCG and FSH receptors has been examined (Campbell et al., “Conversion of Human Choriogonadotropin Into a Follitropin by Protein Engineering,” Proc Natl Acad Sci USA 88(3):760-764 (1991)). The active sites of human FSH have been chemically modified to alter their activity by using an azidobenzoyl derivative of a glycopeptide isolated from the fetuin digest by photoactivation (Rathnam et al., “Conjugation of a Fetuin Glycopeptide to Human Follicle-Stimulating Hormone and its Subunits by Photoactivation,” Biochim Biophys Acta 624(2):436-442 (1980)). Chemical reactions used for the addition, deletion, or replacement of functional epitopes have been fraught with non-specific side reactions and changes in the conformation and function of the proteins. However, the availability of nucleic acid sequences of expressed proteins has opened new avenues to synthesize nucleic acid constructs to express hybrids of two different proteins. This is achieved by the use of site-specific restriction enzymes and ligases to produce chimeric genes, which, in turn, can express the corresponding hybrid proteins. A single-chain construct containing hCG α and β subunits in tandem was fused with the N-terminus of receptor LH-R to investigate their structure-function relationship (Wu et al., “Protein Engineering of Novel Constitutively Active Hormone-Receptor Complex,” J Biol Chem 271:31638-31642 (1996)).
What is needed now is a chimeric nucleic acid molecule that encodes a fusion protein having both a lutropin hormone receptor protein, or a fragment thereof, and a human chorionic gonadotropin protein or a fragment thereof, where the lutropin hormone receptor protein or fragment, and the human chorionic gonadotropin protein or fragment thereof, are immunogenic. Such a bifunctional, immunogenic protein would provide a unique antigen for immunocontraception and gonadal regulation in vertebrates, including humans. The present invention is directed to overcoming these and other deficiencies in the art.